• Journal of Cancer Prevention
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• 제21권3호
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• pp.135-143
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• 2016

Background: Fraxetin (7,8-dihydroxy-6-methoxy coumarin), a coumarin derivative, has been reported to possess antioxidative, anti-inflammatory and neuroprotective effects. A number of recent observations suggest that the induction of heme oxygenase-1 (HO-1) inhibits inflammation and tumorigenesis. In the present study, we determined the effect of fraxetin on HO-1 expression in HaCaT human keratinocytes and investigated its underlying molecular mechanisms. Methods: Reverse transcriptase-PCR and Western blot analysis were performed to detect HO-1 mRNA and protein expression, respectively. Cell viability was measured by the MTS test. The induction of intracellular reactive oxygen species (ROS) by fraxetin was evaluated by 2′,7′-dichlorofluorescin diacetate staining. Results: Fraxetin upregulated mRNA and protein expression of HO-1. Incubation with fraxetin induced the localization of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus and increased the antioxidant response element-reporter gene activity. Fraxetin also induced the phosphorylation of Akt and AMP-activated protein kinase $(AMPK){\alpha}$ and diminished the expression of phosphatase and tensin homolog, a negative regulator of Akt. Pharmacological inhibition of Akt and $AMPK{\alpha}$ abrogated fraxetin-induced expression of HO-1 and nuclear localization of Nrf2. Furthermore, fraxetin generated ROS in a concentration-dependent manner. Conclusions: Fraxetin induces HO-1 expression through activation of Akt/Nrf2 or $AMPK{\alpha}/Nrf2$ pathway in HaCaT cells.

• 대한한의학방제학회지
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• 제31권2호
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• pp.111-124
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• 2023

Objectives : Oxidative stress is an important cause of many diseases including liver injury. Therefore, adequate regulation of oxidative stress plays a pivotal role in maintaining liver function. Until recently, there has been no studies on the hepatoprotective effect of Samchulgeonbi-tang (SCGBT). Therefore, the hepatoprotective effect of SCGBT was investigated in HepG2 cells. In this study, oxidative stress was induced by arachidonic acid (AA) and iron. Methods : To analyze the hepatoprotective effects of SCGBT against oxidative stress induced by AA + iron, the cell viability, apoptosis-related proteins and intracellular ROS, glutathione (GSH), and mitochondrial membrane permeability (MMP) were measured. In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) transcription activation and expressions of Nrf2 target gene were analyzed through immunoblot analysis. Results : SCGBT increased the cell viability from AA + iron - induced cell death and inhibited apoptosis by regulating apoptosis related proteins. SCGBT protected cells by inhibiting ROS production, GSH depletion, and MMP degradation against AA + iron induced oxidative stress. Furthermore, Nrf2 activation was increased by SCGBT, and the Nrf2 target genes were also activated by SCGBT. Conclusions : These results suggest that the SCGBT has a hepatocyte protection effect and antioxidant effect from AA + iron induced oxidative stress.

• The Korean Journal of Physiology and Pharmacology
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• 제24권6호
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• pp.481-492
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• 2020

The present study aimed to examine the effect of allyl isothiocyanate (AITC) on chronic obstructive pulmonary disease and to investigate whether upregulation of multidrug resistance-associated protein 1 (MRP1) associated with the activation of the PARK7 (DJ-1)/nuclear factor erythroid 2-related factor 2 (Nrf2) axis. Lung function indexes and histopathological changes in mice were assessed by lung function detection and H&E staining. The expression levels of Nrf2, MRP1, heme oxygenase-1 (HO-1), and DJ-1 were determined by immunohistochemistry, Western blotting and reverse transcription-quantitative polymerase chain reaction. Next, the expression of DJ-1 in human bronchial epithelial (16HBE) cells was silenced by siRNA, and the effect of DJ-1 expression level on cigarette smoke extract (CSE)-stimulated protein degradation and AITC-induced protein expression was examined. The expression of DJ-1, Nrf2, HO-1, and MRP1 was significantly decreased in the wild type model group, while the expression of each protein was significantly increased after administration of AITC. Silencing the expression of DJ-1 in 16HBE cells accelerated CSE-induced protein degradation, and significantly attenuated the AITC-induced mRNA and protein expression of Nrf2 and MRP1. The present study describes a novel mechanism by which AITC induces MRP1 expression by protecting against CS/CSE-mediated DJ-1 protein degradation via activation of the DJ-1/Nrf2 axis.

• 동의생리병리학회지
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• 제27권6호
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• pp.745-751
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• 2013

Safflower (Carthamus tinctorius L.) seeds have been used in Korea and China for promoting bone formation and protection. This study was designed to examine the Nrf-2 mediated anti-oxidative effects of Korean and Chinese safflower seeds. Water and ethanol extracts of safflower seeds were treated to RAW 264.7 cells. Nrf-2 transcriptional activity was measured by reporter gene assay and western blot analysis. Semi-quantitive RT-PCR analysis was adopted to measure Nrf-2 dependent gene expressions. Water extracts of safflower seeds have strongly induced the activation of Nrf-2 transcription than ethanol extracts. Especially, water extracts of Korean safflower seeds has more strongly increased the expression of nuclear Nrf-2. Water extracts of Korea and China safflower seeds have also increased the expression of Nrf-2-dependent genes such as GCLC, NQO-1 and HO-1 in RAW 264.7 cells. However, all kinds of safflower seeds extracts did not increase intracellular ROS production. These results demonstrate that the antioxidant effects of safflower seeds are not related with ROS production, rather it is mediated by the direct activation of Nrf-2.

• Journal of Ginseng Research
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• 제46권4호
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• pp.550-560
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• 2022

Background: The effect of ginsenoside Rh2 (G-Rh2) on mast cell-mediated anaphylaxis remains unclear. Herein, we investigated the effects of G-Rh2 on OVA-induced asthmatic mice and on mast cell-mediated anaphylaxis. Methods: Asthma model was established for evaluating airway changes and ear allergy. RPMCs and RBL-2H3 were used for in vitro experiments. Calcium uptake, histamine release and degranulation were detected. ELISA and Western blot measured cytokine and protein levels, respectively. Results: G-Rh2 inhibited OVA-induced airway remodeling, the production of TNF-α, IL-4, IL-8, IL-1β and the degranulation of mast cells of asthmatic mice. G-Rh2 inhibited the activation of Syk and Lyn in lung tissue of OVA-induced asthmatic mice. G-Rh2 inhibited serum IgE production in OVA induced asthmatic mice. Furthermore, G-Rh2 reduced the ear allergy in IgE-sensitized mice. G-Rh2 decreased the ear thickness. In vitro experiments G-Rh2 significantly reduced calcium uptake and inhibited histamine release and degranulation in RPMCs. In addition, G-Rh2 reduced the production of IL-1β, TNF-α, IL-8, and IL-4 in IgE-sensitized RBL-2H3 cells. Interestingly, G-Rh2 was involved in the FcεRI pathway activation of mast cells and the transduction of the Lyn/Syk signaling pathway. G-Rh2 inhibited PI3K activity in a dose-dependent manner. By blocking the antigen-induced phosphorylation of Lyn, Syk, LAT, PLCγ2, PI3K ERK1/2 and Raf-1 expression, G-Rh2 inhibited the NF-κB, AKT-Nrf2, and p38MAPK-Nrf2 pathways. However, G-Rh2 up-regulated Keap-1 expression. Meanwhile, G-Rh2 reduced the levels of p-AKT, p38MAPK and Nrf2 in RBL-2H3 sensitized IgE cells and inhibited NF-κB signaling pathway activation by activating the AKT-Nrf2 and p38MAPK-Nrf2 pathways. Conclusion: G-Rh2 inhibits mast cell-induced allergic inflammation, which might be mediated by the AKT-Nrf2/NF-kB and p38MAPK-Nrf2/NF-κB signaling pathways.

• Molecules and Cells
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• 제41권12호
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• pp.1008-1015
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• 2018

$I{\kappa}B$, a cytoplasmic inhibitor of nuclear factor-${\kappa}B$ ($NF-{\kappa}B$), is reportedly degraded via the proteasome. However, we recently found that long-term incubation with proteasome inhibitors (PIs) such as PS-341 or MG132 induces $I{\kappa}B{\alpha}$ degradation via an alternative pathway, lysosome, which results in $NF-{\kappa}B$ activation and confers resistance to PI-induced lung cancer cell death. To enhance the anti-cancer efficacy of PIs, elucidation of the regulatory mechanism of PI-induced $I{\kappa}B{\alpha}$ degradation is necessary. Here, we demonstrated that PI up-regulates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) via both de novo protein synthesis and Kelch-like ECH-associated protein 1 (KEAP1) degradation, which is responsible for $I{\kappa}B{\alpha}$ degradation via macroautophagy activation. PIs increased the protein level of light chain 3B (LC3B, macroautophagy marker), but not lysosome-associated membrane protein 2a (Lamp2a, the receptor for chaperone-mediated autophagy) in NCI-H157 and A549 lung cancer cells. Pretreatment with macroautophagy inhibitor or knock-down of LC3B blocked PI-induced $I{\kappa}B{\alpha}$ degradation. PIs up-regulated Nrf2 by increasing its transcription and mediating degradation of KEAP1 (cytoplasmic inhibitor of Nrf2). Overexpression of dominant-negative Nrf2, which lacks an N-terminal transactivating domain, or knock-down of Nrf2 suppressed PI-induced LC3B protein expression and subsequent $I{\kappa}B{\alpha}$ degradation. Thus, blocking of the Nrf2 pathway enhanced PI-induced cell death. These findings suggest that Nrf2-driven induction of LC3B plays an essential role in PI-induced activation of the $I{\kappa}B$/$NF-{\kappa}B$ pathway, which attenuates the anti-tumor efficacy of PIs.

• BMB Reports
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• 제45권11호
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• pp.647-652
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• 2012

We previously reported that MSTO-211H cells have a higher capacity to regulate Nrf2 activation in response to changes in the cellular redox environment. To further characterize its biological significance, the response of Nrf2, a transcription factor that regulates ARE-containing genes, on the synergistic cytotoxic effect of clofarabine and resveratrol was investigated in mesothelioma cells. The combination treatment showed a marked growth-inhibitory effect, which was accompanied by suppression of Nrf2 activation and decreased expression of heme oxygenase-1 (HO-1). While transient overexpression of Nrf2 conferred protection against the cytotoxicity caused by their combination, knockdown of Nrf2 expression using siRNA enhanced their cytotoxic effect. Pretreatment with Ly294002, a PI3K inhibitor, augmented the decrease in HO-1 level by their combination, whereas no obvious changes were observed in Nrf2 levels. Altogether, these results suggest that the synergistic cytotoxic effect of clofarabine and resveratrol was mediated, at least in part, through suppression of Nrf2 signaling.

• Asian Pacific Journal of Cancer Prevention
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• 제15권6호
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• pp.2911-2916
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• 2014

Oxaliplatin is a first-line therapy for colorectal cancer, but cancer cell resistance to the drug compromises its efficacy. To explore mechanisms of drug resistance, we treated colorectal cancer cells (HCT116 and SW620) long-term with oxaliplatin and established stable oxaliplatin-resistant lines (HCT116-OX and SW620-OX). Compared with parental cell lines, $IC_{50}$s for various chemotherapeutic agents (oxaliplatin, cisplatin and doxorubicin) were increased in oxaliplatin-resistant cell lines and this was accompanied by activation of nuclear factor erythroid-2 p45-related factor 2 (Nrf2) and NADPH quinone oxidoreductase 1 (NQO1). Furthermore, luteolin inhibited the Nrf2 pathway in oxaliplatin-resistant cell lines in a dose-dependent manner. Luteolin also inhibited Nrf2 target gene [NQO1, heme oxygenase-1 (HO-1) and $GST{\alpha}1/2$] expression and decreased reduced glutathione in wild type mouse small intestinal cells. There was no apparent effect in Nrf2-/- mice. Luteolin combined with other chemotherapeutics had greater anti-cancer activity in resistant cell lines (combined index values below 1), indicating a synergistic effect. Therefore, adaptive activation of Nrf2 may contribute to the development of acquired drug-resistance and luteolin could restore sensitivity of oxaliplatin-resistant cell lines to chemotherapeutic drugs. Inhibition of the Nrf2 pathway may be the mechanism for this restored therapeutic response.

• 동의생리병리학회지
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• 제26권3호
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• pp.344-350
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• 2012

Previously, we showed that Dangguisoo-san (DGSS), an herbal formula that has been traditionally used for the treatment of blood stagnation, is also applicable for inflammatory lung diseases. Activation of Nrf2, an anti-inflammatory transcription factor, and suppression of NF-${\kappa}B$, a pro-inflammatory transcription factor, were suggested as an underlying mechanism. However, the constituents responsible for these activities remain unidentified. To this end, we prepared the water extracts of the 9 constituents of DGSS and tested for their effect on Nrf2 by using an Nrf2-Luciferase reporter cell line and western blot analysis. Results show that Carthamus tinctorius L.(CT), one of the 9 constituents of DGSS, strongly activated Nrf2. Similarly, when measured the effect of the 9 constituents on NF-${\kappa}B$ by using an NF-${\kappa}B$-Luciferase reporter cell line and western blotting for nuclear p65, indicative of activated NF-${\kappa}B$, most constituents were capable of suppressing NF-${\kappa}B$ in various degrees. However, CT and Cyperus rotundus L. (CR) strongly suppressed NF-${\kappa}B$ activity elicited by LPS. Of note, CT activated Nrf2 and suppressed NF-${\kappa}B$ strongly as well. Our results contributes to corroborating the anti-inflammatory effects of DGSS by identifying CT and CR as two major herbs responsible for activating Nrf2 and suppressing NF-${\kappa}B$. These results suggest that CT and CR represent some of the effects of DGSS in the regulation of inflammation.

• Biomolecules & Therapeutics
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• 제20권2호
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• pp.144-151
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• 2012

The molecular mechanisms by which a variety of naturally-occurring dietary compounds exert chemopreventive effects have been a subject of intense scientific investigations. Induction of phase II detoxification and anti-oxidant enzymes through activation of Nrf2/ARE-dependent gene is recognized as one of the major cellular defense mechanisms against oxidative or xenobiotic stresses and currently represents a critical chemopreventive mechanism of action. In the present review, the functional significance of Keap1/Nrf2 protein module in regulating ARE-dependent phase II detoxification and anti-oxidant gene expression is discussed. The biochemical mechanisms underlying the phosphorylation and expression of Keap1/Nrf2 proteins that are controlled by the intracellular signaling kinases and ubiquitin-mediated E3 ligase system as well as control of nucleocytoplasmic translocation of Nrf2 by its innate nuclear export signal (NES) are described.